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Cited by 10Plant tissue culture and regenerationPlant Stress Responses and TolerancePostharvest Quality and Shelf Life Management

Efficient Cryopreservation of Populus tremula by In Vitro-Grown Axillary Buds and Genetic Stability of Recovered Plants

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Vidyagina, Elena O., Kharchenko, Nikolay N., Shestibratov, Konstantin A.·2021·Plants
DOI 10.3390/plants10010077PMC PMC7823548
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Abstract

Axillary buds of in vitro microshoots were successfully frozen at –196 °C by the one-step freezing method using the protective vitrification solution 2 (PVS2). Microshoots were taken from 11 transgenic lines and three wild type lines. Influence of different explant pretreatments were analyzed from the point of their influence towards recovery after cryopreservation. It was found out that the use of axillary buds as explants after removal of the apical one increases recovery on average by 8%. The cultivation on growth medium of higher density insignificantly raises the regenerants survival rate. Pretreatment of the osmotic fluid (OF) shows the greatest influence on the survival rate. It leads to the increase in survival rate by 20%. The cryopreservation technology providing regenerants average survival rate of 83% was developed. It was based on the experimental results obtained with explant pretreatment. Incubation time in liquid nitrogen did not affect the explants survival rate after thawing. After six months cryostorage of samples their genetic variability was analyzed. Six variable simple sequence repeat (SSR) loci were used to analyze genotype variability after the freezing-thawing procedure. The microsatellite analysis showed the genetic status identity of plants after cryopreservation and of the original genotypes. The presence of the recombinant gene in the transgenic lines after cryostorage were confirmed so as the interclonal variation in the growth rate under greenhouse conditions. The developed technique is recommended for long-term storage of various breeding and genetically modified lines of aspen plants, as it provides a high percentage of explants survival with no changes in genotype.

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  • Choice of explant influences genetic stability, with meristematic tissues such as axillary buds, shoot tips, and root tips generally exhibiting higher genetic stability than differentiated tissues like leaves and stems, which are more susceptible to genetic alterations during culture.

    “Additionally, the choice of explant influences genetic stability, with meristematic tissues such as axillary buds [69], shoot tips [66], and root tips [22] generally exhibiting higher genetic stability among regenerated and donor plants compared to differentiated tissues like leaves [70] and stems [71], which are more susceptible to genetic alterations during culture.”

    From Somaclonal Variation and Clonal Fidelity in Commercial Micropropagation: Challenges and Perspectives (2025) · cites this paper as [69]

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  • Cryopreservation has proven to be an effective long-term strategy for preserving plant tissues, embryogenic lines, and somatic embryos at ultra-low temperatures (-196°C) without compromising their genetic integrity.

    “Furthermore, cryopreservation has proven to be an effective long-term strategy for preserving plant tissues, embryogenic lines, and somatic embryos at ultra-low temperatures (-196 • C) without compromising their genetic integrity [69].”

    From Somaclonal Variation and Clonal Fidelity in Commercial Micropropagation: Challenges and Perspectives (2025) · cites this paper as [69]

    The Introduction (page not specified) describes cryopreservation at −196 °C as a promising way to store plant material unchanged for a theoretically unlimited time. The Abstract and Results §2.3 (pages not specified) report genetic identity after six months of cryostorage, but the study tested aspen axillary buds, not embryogenic lines or somatic embryos; it does not establish genetic integrity for those materials. (LLM, conf=0.88)

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Authors
Vidyagina, E. O., Kharchenko, N. N., & Shestibratov, K. A.
Affiliations
  • Russian Academy of Sciences (RU)
  • Voronezh State University of Forestry and Technologies (RU)
Corresponding author
Shestibratov, Konstantin A.
ORCID
Venue
Plants
Publisher
MDPI AG
Published
2021-01-02
Volume
10
Issue
1
Pages
77
ISSN
2223-7747 (ISSN-L)
DOI
10.3390/plants10010077
PMC
PMC7823548
OpenAlex
W3118809528
Cited by
10
References
52
Citations by year
2026: 2 · 2025: 2 · 2024: 1 · 2023: 2 · 2022: 3
FWCI
0.47
Type
journal article
Language
en
Open access
Licence
Locations
  • Plants (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Ministry of Science and High Education of the Russian Federation ("Plant molecular biology and biotechnology: their cultivation, pathogen and stress protection (BIBCH)" (No 0101-2019-0037))
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant tissue culture and regeneration
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Stress Responses and Tolerance
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Postharvest Quality and Shelf Life Management
Keywords
Populus tremula, aspen, cryopreservation, axillary buds, microshoots, genetic stability, transgenic strain, genotypic variation, microsatellite markers, long-term storage, genetic modification

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